CN108486024A - The method of sensor-based system detection organophosphorus pesticide based on flora - Google Patents

Abstract

The method for the sensor-based system detection organophosphorus pesticide that the present invention provides a kind of based on flora, the sensor-based system includes signal induction module containing OPH and the genetic engineering flora containing signal generating module.One of them is used to OPs being hydrolyzed into p-nitrophenol (PNP), and PNP signals are converted to beta galactosidase and generated for colorimetric detection by another.By optimization, which can induce in 3.5 hours at 28 DEG C with 1 × 10^‑9The Concentration Testing ethyl paraoxon of M is about 200 times higher than unicellular full cell sensor sensitivity.In addition, it can detect common a plurality of types of OP in agricultural.In addition, the sensor-based system can be prepared into detection organophosphorus pesticide filter paper, the foreground of portable field detection is illustrated.The present invention provides a sensitivity, quick and portable pollutant monitoring bioanalytical sensing platforms, also show the utility model environmental applications of the engineered microbes ecosystem.

Description

Method for the detection of organophosphorus pesticides by a bacterial community-based sensing system

technical field

The invention relates to the technical field of pesticide detection, in particular to a method for detecting organophosphorus pesticides with a bacterial flora-based sensing system.

Background technique

Organophosphorus pesticides (OPs) are widely used in insect control, accounting for approximately 38% of global pesticide use. As potent inhibitors of acetylcholinesterase in the nervous system, they can also cause acute neurotoxic poisoning in humans and animals. Therefore, despite the substantial agricultural benefits of OPs, the large-scale application of OPs raises serious public concerns about health, environment, and food safety. Therefore, there is an urgent need to develop methods for rapid, sensitive and portable OPs detection. Many analytical methods, such as liquid chromatography or gas chromatography and mass spectrometry, have been developed for the detection of OPs. However, they are often expensive and require extensive logistics. In recent years, biosensors have attracted increasing attention due to their simple operation, fast response, and high cost performance. For OPs, organophosphate hydrolase (OPH), a highly efficient OPs-converting enzyme, has been immobilized on the surface of an electrochemical analytical detector, providing an alternative method for the detection of OPs compounds. However, this type of sensor is often interfered by other oxidizable species such as glucose, sucrose, and phenol in real samples.

A promising approach to address the limitations of existing approaches is whole-cell biosensing, which exploits distinct regulatory elements of microorganisms to detect environmental signals. Whole-cell biosensors typically consist of a transcriptional regulator that binds its target analyte and a reporter gene that translates the regulator-target interaction into a measurable output. For OPs detection, Chong et al. constructed a novel whole-cell Escherichia coli biosensor by detecting and reporting a p-nitrophenol (PNP) in OPs hydrolyzate with dimethylphenol regulatory protein (DmpR). Notably, since DmpR is not a natural regulator of PNP sensitivity, the researchers employed a systematic engineering approach to optimize DmpR, resulting in detection sensitivity. However, the sensitivity is still orders of magnitude lower than electrochemical and chromatographic methods. Also, it takes a long time to respond (over 6 hours).

As part of the PNP degradation pathway from Pseudomonas, strain WBC-3, the LysR-type transcriptional regulator LTTR (denoted PnpR) has been shown to activate three operons (pnpA, pnpB and pnpCDEFG) in response to PNP. Therefore, a promising solution to utilize this native PNP response regulator can be a promising solution for the development of ultrasensitive OPs biosensors. In addition to the catalytic efficiency of OPHs, PNP sensing also largely depends on the physical accessibility of OPHs to OPHs. Studies have shown that the diffusion of OPs in the cytoplasm where OPH resides is a rate-limiting step in PNP induction. In fact, OPH's activity increased sevenfold when it was anchored to the outer membrane. One problem associated with surface display is that prolonged incubations require proper target protein translocation, leading to delays and non-linear signal accumulation.

Recently, synthetic microbial consortia have emerged as an effective way to program cellular functions. A range of man-made ecosystems have been successfully created, producing large quantities of chemicals and biomacromolecules and generating defined population dynamics. These well-designed ecosystems have several distinct advantages over single engineered strains, including enhanced performance, stability, and programmability of cellular functions through the division of labor among species within the ecosystem.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for detecting organophosphorus pesticides with a bacterial flora-based sensing system for deficiencies in the prior art.

The purpose of the present invention is achieved through the following technical solutions:

In the first aspect, the present invention provides a bacterial flora-based sensing system, including a genetically engineered flora containing an OPH signal sensing module and a signal generating module.

Preferably, the construction method of the genetically engineered bacterium containing the OPH signal sensing module comprises the following steps: connecting the OPH with the pVLT33 carrier, constructing the pPNCO33 plasmid, and then transforming the pPNCO33 plasmid into the genetically engineered bacterium to form a bacterium containing the OPH signal sensing module. Genetically engineered bacteria. Preferably, the genetically engineered bacterium is Escherichia coli XL1-Blue.

Preferably, the construction of the genetically engineered bacteria containing the signal generation module comprises the following steps:

The pnpR-PpnpC gene sequence was inserted into the pSV-β-galactosidase vector by overlapping extension polymerase chain reaction to form a pSVRTCL plasmid, and the expression of the pnpR gene introduced into the constructed plasmid was regulated by the constitutive promoter SV40, and the introduced pnpC was activated The pSVRTCL plasmid is responsible for regulating the expression of the downstream lacZ gene; then the pSVRTCL plasmid is transformed into Escherichia coli to form a genetically engineered bacterium containing a signal generation module. Preferably, the genetically engineered bacteria is Escherichia coli DH5α.

Preferably, the relative ratio of the genetically engineered bacteria containing the OPH signal sensing module to the genetically engineered bacteria containing the signal generating module is 10:1˜1:10.

Preferably, the total densities of the genetically engineered bacteria containing the OPH signal sensing module and the genetically engineered bacteria containing the signal generating module are the same.

In a second aspect, the present invention provides an application of the aforementioned sensing system in the detection of organophosphorus pesticides.

Preferably, the organophosphorus pesticides include ethyl paraoxon, methyl paraoxon, ethyl parathion, methyl parathion, ethyl p-nitrophenylphenylphosphorothioate (EPN) and borer pine.

In a third aspect, the present invention provides a method for detecting organophosphorus pesticides with a flora-based sensing system, comprising the following steps:

The aforementioned sensing system was suspended in 2YT liquid medium containing X-gal and organophosphate, and co-incubated at 28°C, and the concentration of organophosphate was measured by color intensity.

In a fourth aspect, the present invention provides a filter paper for detecting organophosphorus pesticides based on the aforementioned sensing system.

In a fifth aspect, the present invention provides a method for preparing filter paper for detecting organophosphorus pesticides, comprising the following steps: suspending the aforementioned genetically engineered bacteria containing the OPH signal sensing module and the genetically engineered bacteria containing the signal generating module in the drying protectant solution, and drop it on the filter paper strip under sterile conditions to form a cell area, and then vacuum freeze-dry the filter paper strip to obtain the obtained product.

In a sixth aspect, the present invention provides a method for using filter paper for detecting organophosphorus pesticides, comprising the following steps: dissolving the solution to be tested containing organophosphate in 2YT liquid medium, and then inserting the cell region of the filter paper into the medium After incubation at 28°C for 2 hours, X-gal was added to the cell area of the filter paper, and the concentration of organophosphorus was determined by color intensity.

Compared with the prior art, the present invention has the following beneficial effects:

1. The flora-based sensing system of the present invention can be used to detect and respond to OPs, and the system has a lower detection limit and high sensitivity at nanomolar concentrations. The lowest detection limit can reach 1×10 ^-9 M.

2. Compared with a single engineered strain whose transformation and detection steps are usually difficult to change, the colony-based implementation of OPs sensing allows simple and efficient adjustment between transformation and detection by simply changing the relative population abundance.

3. The system can work in both liquid and freeze-dried states, and has wide applicability.

4. Its rapid colorimetric response to OPs eliminates the need for sophisticated instruments, which provides a feasible analytical test in low-resource settings.

5. Although the biosensing system is designed to detect PNP converted from OPs, it can in principle be used to detect PNP from any other source (47). Therefore, the system can be used as an independent subsystem in other PNP-related fields.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Figure 1: Schematic diagram of the bacterial flora-based biosensing system used for OPs detection in the present invention;

Figure 2: The plasmid map of pPNCO33 and the results of whole-cell OPH activity in the examples of the present invention; wherein Figure 2A is the plasmid map of pPNCO33; Figure 2B is the result of OPH activity of whole cells;

Figure 3: The maps of the pBBR1-tacpnpR plasmid and pCMgfp-spClacZ plasmid and the dose response of the signal generation module to the PNP concentration in the embodiment of the present invention; wherein Figure 3A is the map of the pBBR1-tacpnpR plasmid and the pCMgfp-spClacZ plasmid; Figure 3B is the signal The dose response of the generation module to the PNP concentration;

Fig. 4: In the embodiment of the present invention, ethyl paraoxon is used to test the results of the biosensing system based on flora; wherein, Fig. 4A is two kinds of Escherichia coli containing signal generation modules; Fig. 4B is the presence of different ethyl paraoxon The absorbance at 650nm of the culture suspension of the concentration; Figure 4C is the picture of the colorimetric test of two different systems under different concentrations of ethyl-paraoxon;

Figure 5: The absorbance of biosensor samples corresponding to different ethyl paraoxon inoculation concentration time functions in the embodiment of the present invention;

Figure 6: The results of detecting different OPs using a system based on flora; wherein Figure 6A is the structure of the OPs to be detected; Figure 6B is the detection result;

Figure 7: The result of detecting ethyl paraoxon with paper based on the flora sensing system; wherein Figure 7A is an image of the colorimetric result obtained with a digital camera; Figure 7B is the color intensity using the results in Figure 7A in B Plotting the function of base paraoxon concentration;

Figure 8: The colorimetric test results of the flora sensing system using actual samples;

Figure 9: Absorbance results of the colony-based sensing system using different ratio mixing;

Figure 10: Plasmid maps of pSVRCL and pSVRTCL; wherein Figure 10A is the pSVRCL plasmid; Figure 10B is the pSVRTCL plasmid;

Figure 11: Colorimetric response to different concentrations of ethyl paraoxon by using a colony sensing system containing signal-generating cells carrying pSVRCL plasmid;

Figure 12: Time course of growth of E. coli carrying pSVRTCL and pBBR1-tacpnpR;

Figure 13: Monitoring of β-galactosidase expression using chemiluminescence experiments; where Figure 13A is a diagram of the light generation mechanism in the presence of β-galactosidase; Figure 13B is the β-galactosidase in samples with different concentrations of OPs based on chemiluminescence measurement Calibration curve for galactosidase expression.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Example 1

1. The bacterial strains, medium and reagents used in this embodiment

Escherichia coli XL1-Blue (Clontech, USA) and Escherichia coli DH5α (Invitrogen, USA) were used for all cloning and protein expression steps. Bacteria were cultured in 2YT medium (16 g/L tryptone, 10 g/L yeast extract and 5 g/L sodium chloride).

Dissolve p-nitrophenol (PNP) and Ops (such as ethyl paraoxon, methyl paraoxon, ethyl parathion, methyl parathion, fenitrothion and EPN) in acetonitrile to prepare concentrations It is 1×10 ^-1 M stock solution.

Test chemicals were purchased from Sigma-Aldrich Company. Whatman Universal filter paper (medium speed, creped, diameter = 9.0 cm), animal tissue peptone, beef extract, gelatin, L-sodium ascorbate, D-(+)-sodium pentafluoride, L-glutamic acid monosodium salt Hydrate, ampicillin, kanamycin, tetracycline and anhydrous N,N-dimethylformamide (DMF) were purchased from Promega (Madison, WI, USA). HPLC grade acetonitrile, sodium chloride, potassium chloride, magnesium chloride, sodium phosphate monohydrate and disodium phosphate heptahydrate were purchased from Fisher Scientific (Pittsburgh, PA, USA). The chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) was purchased from Gold Biotechnology (St. Louis, MO, USA). β-Glo assay reagents were obtained from Promega (Madison, WI, USA). Alpha 2-4 lyophilizers were purchased from Martin Christ Gefriertrocknungsanlagen GmbH (Osterode, Germany). Polymerase chain reaction (PCR) was performed using Q5 high-fidelity DNA polymerase or Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA) and QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA), and recombinant plasmids were recovered using QIA prep Spin Miniprep Kit (Qiagen, Valencia, CA, USA).

2. The construction of the bacterial colony-based sensing system

2.1 Construction of Escherichia coli containing OPH signal sensing module

The OPH signal-sensing module contains the inpnc-opd fragment (encoded INPNC-OPH fusion protein) under the control of the IPTG-inducible tac promoter. This was placed in the pVLT33-based vector pPNCO33 (Fig. 2A). The opd portion encodes a hydrolase that converts OP to PNP. The inpnc-opd fragment of the inpnc-encoded truncated version of INP, which contains only its N- and C-terminal domains, has been widely used as a surface anchoring motif. This fusion design allows OPH to be displayed on the cell surface to increase the OP-PNP turnover rate. Because the overexpression of active OPH on the cell surface is highly host-specific and exhibited a higher rate of OP degradation in the XL1-Blue strain, the pPNCO33 plasmid was transformed into E. coli XL1-Blue.

A fragment encoding the INPNC-OPH organophosphate hydrolase was PCR amplified from the pINCOP plasmid and cloned into EcoRI-HindIII digested pVLT33 (an E. coli-Pseudomonas shuttle vector) to generate pPNCO33. The pPNCO33 plasmid was transformed into Escherichia coli XL1-Blue to form the OPH signal sensing module, and E. coli containing the OPH signal sensing module was obtained.

The Escherichia coli carrying the pPNCO33 plasmid was cultured with shaking at 250 rpm and 37° C. in 2YT medium containing kanamycin (50 μg/mL). When the OD600 of the E. coli culture reached 0.6, 0.3 mM IPTG was added and protein expression was induced at 28°C for 24 hours.

2.2 Escherichia coli containing signal generation module

To construct the pCMgfp-spClacZ plasmid, the mcs-gfp fragment (where mcs is the multiple cloning site) was amplified from the pEX18Tc-cmgfp template and cloned into KpnI-SacI digested pCM130 to obtain the pCMgfp plasmid. The lacZ fragment was cloned into BamHI-HindIII digested pCMgfp to generate the pCMgfp-lacZ plasmid. The pnpC promoter (-94 to +25 region) was cloned into pCMgfp-lacZ digested with SphI-BamHI to generate the pCMgfp-spClacZ plasmid (shown in Figure 3A) in which the lacZ gene was located on the pnpC promoter downstream.

The tac fragment was digested with KpnI and BamHI, the pnpR fragment containing the His tag sequence was digested with BamHI and SacI, and these two fragments were cloned into pBBR1mcs-2 digested with KpnI-SacI to obtain the pBBR1-tacpnpR plasmid.

The pCMgfp-spClacZ plasmid and the pBBR1-tacpnpR plasmid were co-transformed into a single E. coli DH5α cell to form a signaling module (ie, E. coli 1 containing a signaling module). Transformed Escherichia coli DH5α was cultured with shaking in 2YT medium supplemented with kanamycin (50 μg/mL) and tetracycline (100 μg/mL) at 250 rpm and 37°C. Once the OD ₆₀₀ of the E. coli culture reaches 0.6, add 0.5mM IPTG to it to express the PnpR protein, which combines with the PNP produced by the OPH module to sense organophosphorus pesticides, and stimulates the expression of β-galactosidase, and the catalytic signal indicates the molecule X-gal Decomposition produces a blue signal.

To test the function of the building block E. coli 1 containing the signal-generating module, the signal-generating E. coli DH5α cells harboring pCMgfp-spClacZ and pBBR1-tacpnpR were grown to an _OD600 of 1 in 100-mL culture flasks, and then 90 μL of Aliquot into 96-well microtiter plates. After adding different concentrations of PNP, 10 μL of 10 mg/mL X-gal solution was added to each well, and the culture plate was incubated at 28° C. with shaking at 220 rpm. The absorbance at 650 nm was measured every 30 minutes using a SpectraMax○R M5 microplate reader for a total of 3.5 hours.

PNP binds to the corresponding pnpC promoter to induce the expression of the downstream b-galactase gene, and decomposes X-gal to produce blue. The response of the system to PNP was determined by quantifying the blue color produced by the reporter protein β-galactosidase after X-gal depletion, which can be achieved by measuring the absorbance at 650 nm. Indeed, the data clearly show that the absorbance of the blue color at 650 nm is positively correlated with the PNP concentration (Fig. 3B).

The promoters of the wild-type pnpR and the PNP catabolic operon pnpC were amplified from the pBBR1-tacpnpR and pCMgfp-spClacZ plasmids, respectively, and used to construct individual plasmids producing signals (primer lists are shown in Table 1). Amplified products were separated and purified by gel electrophoresis. The two genes, pnpR and pnpC, were fused by overlap extension PCR (OEP). The pSV-β-galactosidase plasmid vector (pSV-β-galactosidase vector) was purchased from Promega (Madison, WI, USA). The pSV-β-galactosidase vector and fused pnpR-pnpC were double digested with HindIII-BsaAI. Digests of pSV-β-galactosidase and fused pnpR-pnpC were mixed together and ligated overnight at 16°C to generate pSVRCL plasmid (Fig. 10A). In this plasmid, the pnpR gene was expressed under the control of the constitutive promoter SV40, and the lacZ gene was expressed under the control of the pnpC promoter. The pSVRCL plasmid was transformed into E. coli DH5α to form E. coli 2 containing the signal generation module.

Table 1

E. coli 2 containing the signaling module was subjected to a microtiter plate assay to test the response of the system. Unexpectedly, the system lost the dose-dependent response, as a distinct blue color appeared after 2 hours of incubation with and without paraoxon (Figure 11).

To terminate the activity of the SV40 promoter at the desired position, the long T7 terminal region (48bp, T7 terminator) from the pET-28a(+) plasmid was inserted between pnpR and pnpC to generate the pSVRTCL plasmid. The recombinant plasmid pSVRTCL was transformed into Escherichia coli DH5α to form Escherichia coli 3 containing the signal generation module. E. coli 3 containing the signaling module was subjected to a microtiter plate assay to test the response of the system. As shown in Figure 4A and Figure 10B, introduction of the T7 terminator restored the previously observed concentration-dependent response. To test whether increased amounts of regulatory proteins inhibit cell growth, we compared the growth kinetics of cells harboring pSVRTCL and pBBR1-tacpnpR plasmids. Escherichia coli carrying pSVRTCL and pBBR1-tacpnpR was cultured at 37°C for several hours in 2YT medium containing 100 µg/mL ampicillin and 50 µg/mL kanamycin. When the _OD600 of the bacterial culture containing pBBR1-tacpnpR reached 0.6, 0.5 mM IPTG was added and both cell cultures were incubated at 30 °C for an additional 24 h. Cell concentration was determined by measuring the _OD600 of the culture medium. The results showed that increased production of PnpR did not lead to significant growth inhibition (Figure 12).

The β-galactosidase activity of Escherichia coli 3 containing the signal generation module was detected by chemiluminescence assay. Specific steps are as follows:

Pick a fresh single colony of Escherichia coli 3 containing the signal generating module from the plate, and culture it in a test tube containing 5 mL of 2YT medium. After culturing for 6 hours, 1 mL of the culture solution was transferred to a culture flask containing 100-mL of 2YT medium. When the culture reached an _OD600 of 1, 90 μL of pSVRTCL cell culture was added to a 96-well plate containing 90 μL of OPH cell culture ( _OD600 = 1) expressed for 24 hours. Finally, 20 μL of different concentrations of OP was added to each well, and the 96-well plate was placed in a shaker at 28 °C at 220 rpm. After 3.5 hours, the cell culture was centrifuged and the cells were resuspended in 200 μL of fresh 2YT medium to remove OPs compounds. Finally, cells with an _OD600 of 0.30 were collected and placed in a black 96-well plate for β-galactosidase activity assay. The expression level of the reporter protein (β-galactosidase) was determined using a chemiluminescence-based β-galactosidase assay system, and the operation method was referred to the product manual. Induced chemiluminescence (1 s/well) was measured. The results are shown in 3B, the light absorption is positively correlated with the PNP concentration.

3. Whole cell OPH activity

Whole-cell OPH activity was measured using paraoxon ethyl as a substrate. E. coli cells expressing recombinant OPH were harvested after 6, 12, 18 and 24 hours of IPTG induction, washed twice with 100 mM phosphate buffer (pH 7.4), and then resuspended in the same buffer to an _OD600 of 1 . Hydrolysis of ethylparaoxon was measured by spectrophotometrically measuring PNP formation at 405 nm (ε 405 = 16,600 M ⁻¹ cm ⁻¹ ). OPH activity assays were performed at 28°C in 100 mM phosphate buffer (pH 7.4) supplemented with 1×10 ⁻⁴ M substrate and 100 μL of cells (OD ₆₀₀ =1.0). Enzyme activity is represented by the enzyme activity unit of 1.0OD ₆₀₀ whole cells (1 enzyme activity unit is equal to the hydrolysis of 1 micromole of substrate per minute). As a result, as shown in Figure 2B, the OPH catalytic activity started to appear in the exponential phase and continued to increase after the cells reached the stationary phase. Notably, this time course relationship indicates the time required for complete transfer of the INPNC-OPH fusion to the cell surface. Therefore, cells at the late stage of stabilization (24 hours) were used for OP degradation, as these cells can efficiently decompose pollutants and produce the greatest amount of PNP for the signaling module.

4. Signal response of flora biosensing system to OPs

The schematic diagram of the bacterium-based biosensing system for OPs detection is shown in Fig. 1. Dissolve commercially available OPs such as ethyl paraoxon, methyl paraoxon, ethyl parathion, methyl parathion, EPN and fenitrothion in acetonitrile to obtain a concentration of 1 × ^10-1 M Reserve solution.

The OPH E. coli cells that had been cultured for 24 hours (that is, E. coli containing the OPH signal sensing module) were collected by centrifugation, and diluted with fresh medium to achieve an _OD600 of 1.

The three signal-generating E. coli cells (ie, E. coli 1-3 containing the signal-generating module) were all grown in 100-mL culture flasks until the OD ₆₀₀ was 1.

A 1:1 ratio of OPH E. coli to generator E. coli cells is used for 96-well microtiter plate-based assays of OPs. 20 μL of stock solutions of different OPs compounds were used to dilute to obtain a final concentration of 1×10 ⁻⁴ ~1×10 ⁻¹⁰ M. 10 μL of 10 mg/mL X-gal solution was added to each well, and then the culture plate was incubated on a plate shaker at 220 rpm and 28°C. Aliquots induced with OP were incubated for a maximum of 5 hours and the absorbance at 650 nm of induced and uninduced cultures was measured at 30 min intervals using a SpectraMax® M5 microplate reader. As shown in Figure 5, absorbance values increase with increasing sensing (or exposure) time, as reflected by the positive slope of the curve. As shown in Figure 5B, the system exhibited a predictable and dose-dependent manner, consistent with the results of visual inspection. As shown in Figure 6A, we found that, similar to the signal response observed in the ethyl paraoxon assay, the color of the co-culture medium gradually changed from light yellow to dark blue for all pesticides tested (Figure 6B). For the OPs tested, we also performed a chemiluminescence-based assay by measuring the extent of the enzymatic reaction (Figure 13). By comparing the detection results, it was found that for different pesticides, the sensitivities based on the flora system ranged from 1×10 ^-9 M to 1×10 ^-5 M. For common OPs compounds, the system is 2-4 times more sensitive than single-cell colorimetric methods. We also noted that the fenitrothion sensitivity was 1×10 ^-5 M, i.e. this could be due to inefficient catalytic performance of OPH on this substrate or due to the formation of 2-methyl-4-nitrophenol instead of p-nitro Phenol production.

By changing the relative abundance (10:1, 3:1, 1:1, 1:3 and 1:10) of the two cell types (E. coli with OPH signal sensing module and E. coli 1 with signal generation module) , while keeping their overall densities the same (OD600 = 1.0), we found that the output color absorbance followed a “bell-shaped” variation, with the highest absorbance occurring at the 1:1 and 1:3 ratios (Fig. 9). This result confirms three layers of information. First, the flora-based system was able to successfully detect ethyl paraoxon. Second, the assay requires two strains for the task, namely OPH conversion and signal generation. Third, the performance of the assay depends on the relative abundance of the two strains. At a 1:1 ratio, we next co-cultured suspension cultures of the two strains by supplementing 2YT liquid medium with different concentrations of paraoxon ethyl (from 1×10 ⁻¹⁰ to 1×10 ⁻⁶ ). The detection limit of the cultured biosensing system was 10 ^-4 M. As shown in Figure 4C (row I), we observed that the color intensity of the responses increased with the concentration of ethyl paraoxon, indicating a positive correlation between the color intensity and the concentration of ethyl paraoxon. Notably, the limit of system response was found to be 1 x 10 ^-6 M ethyl paraoxon. From this assay, we also found that cellular coupling of fully active surface-expressed OPH (which more readily reaches the threshold amount of PNP) to a signal in the exponential growth phase resulted in a significantly shorter response time (3.5 h) than previously reported ( about 6 hours). Ethyl paraoxon concentrations as low as 1 x 10 ^-9 M also showed a marked increase in color compared to the control. In contrast, at concentrations higher than 1×10 ^-6 M, a signal was detectable within 2 hours.

Co-cultivation of OPH Escherichia coli with suspension cultures of Escherichia coli 2 or 3 containing the signal generating module, respectively, corresponds to the sensitivity to ethyl paraoxon, the structure is shown in Figure 4A and Figure ^4B . Detectable levels of color were also observed at concentrations of ethyl paraoxon (FIG. 4C, row II). The results showed that the sensitivity of E. coli 2 or 3 containing the signaling module was successfully increased by three orders of magnitude from the starting system (cells containing pBBR1-tacpnpR and pCMgfp-spClacZ plasmids) (Fig. 4A, row I; Fig. 4B, circles and Figure 4C, first row).

Embodiment 2, the preparation of filter paper sensing strip

Sensing paper strips were prepared according to the aforementioned flora-based biosensing system. Briefly, the two cell types were suspended in a 1:1 ratio in sterile dry protection solution pre-warmed at 37°C. 5 μL of the cell suspension was spotted on Whatman filter paper strips (0.6×4 cm), and the strips were dried in a laminar air oven for 10 minutes, followed by vacuum drying at 20° C. in a lyophilizer. The filter paper sensor strips were then stored at 4°C until use.

Embodiment 3, the mensuration of the dose-response OPs curve on the filter paper sensing strip

Ethyl paraoxon (concentration of 1×10 - ³ -1×10 ^-9 M) was diluted with HEPES buffer (pH 7.0) containing 50 mM each of sodium chloride, potassium chloride and magnesium chloride. Take 100 μL of each standard solution and add to the culture tube containing 900 μL 2YT medium and prepared filter paper strips. The culture tubes were then incubated statically at 28°C for 2 hours. The paper strips were then removed from the culture tubes and kept between layers of plastic wrap to prevent drying. Subsequently, 10 μL of X-gal substrate solution (50 mg/mL) dissolved in DMF was carefully added to the site containing the sensor cells, and color development was allowed to proceed for 90 minutes. As shown in Figure 7A, as the concentration of ethyl paraoxon was increased, the cell-retained area of the paper strip changed from colorless to blue.

In addition to visual observation of the developed blue color, color intensity was also measured using ImageJ software (NIH, Bethesda, MD, USA) (http://rsbweb.nih.gov/ij/). Briefly, measurements in ImageJ were set to mean gray values and images were inverted. The inversion of the image was necessary because ImageJ recorded 100% black as 0 and 100% white as 255. Use the selection tool to draw a rectangular section around the point to be measured and record the average gray value. The area of the same rectangle around the light spot is determined as the background value to account for variations in color intensity due to differences in ambient lighting when the photo was taken. Basically, the final color intensity signal is corrected for blank and illuminance dependent color intensity variations. ImageJ software analysis confirmed the results obtained by visual evaluation of the strips (Figure 7B), showing a linear relationship between the measured color intensity and the logarithm of the ethylparaoxon concentration. The results also showed that under this setting, the sensing system could clearly detect ethyl paraoxon at a concentration of 1×10 ^-7 M within 1.5 h. Taken together, these results demonstrate the feasibility of using paper-based flora biosensors to reliably detect OPs, which is particularly useful in developing countries and remote societies.

Embodiment 4, real sample test

This example prepares and measures apple and soil samples to which paraoxon ethyl has been added. In this assay, approximately 1 g of soil was collected and dissolved in 900 μl of water. For comparison, a standard solution of ethyl paraoxon (1×10 ⁻⁵ or 1×10 ⁻⁶ M) was also tested. The collected apples were cut into pieces and mashed, and 1 g of the crushed apples was dissolved in 900 μl of water to form sample solution 1. 1 g of the collected soil was dissolved in 900 μl of water to form sample solution 2.

The two sample solutions were centrifuged at 37 °C and 8000 rpm for 10 min, and the supernatant was filtered through a 0.22-μm PVDF filter and diluted 2-fold with deionized water. Subsequently, the supernatant was added to the ethyl paraoxon solution to obtain concentrations of 1×10 ^-5 and 1×10 ^-6 M. Finally, the concentration of the added standard OPs was detected using a developed biosensor and the absorbance at 650 nm was measured using a Spectramax® RM5 plate reader. As shown in Figure 8, the responses obtained for the apple and soil samples followed the same pattern as the standard solutions. In addition, the recoveries of spiked ethyl paraoxon in apple and soil samples ranged from 89.40 ± 5.1 to 94.80 ± 6.9% (Table 2), indicating that the novel sensing platform can be applied to the practical determination of OPs samples.

The recovery rate of standard addition of ethyl paraoxon in table 2 apple and soil (value is the mean value of three determinations)

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the case of no conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

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<211> 33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgggatccca tgatttcacc ttttttgttg taa 33

<210> 5

<211> 29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgaagcttat ggcgccaggc gtggtattt 29

<210> 6

<211> 40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

accatcatca tcatcattaa ctgtgctgtt gatcgttcgc 40

<210> 7

<211> 40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcgaacgatc aacagcacag ttaatgatga tgatgatggt 40

<210> 8

<211> 33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgcacgtaca tgatttcacc ttttttgttg taa 33

<210> 9

<211> 29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgaagcttat ggcgccaggc gtggtattt 29

<210> 10

<211> 68

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttggc ctgtgctgtt 60

gatcgttc68

<210> 11

<211> 43

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gccccaaggg gttatgcatg ttaatgatga tgatgatggt gtt 43

<210> 12

<211> 33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcacgtaca tgatttcacc ttttttgttg taa 33

Claims (10)

1. a kind of sensor-based system based on flora, which is characterized in that including containing OPH signal induction modules and containing signal generating module Genetic engineering flora.

2. the sensor-based system according to claim 1 based on flora, which is characterized in that the signal induction module containing OPH The construction method of genetic engineering bacterium include the following steps：OPH and pVLT33 carriers are attached, pPNCO33 plasmids are built, Then pPNCO33 plasmids are transformed into the genetic engineering bacterium that the signal induction module containing OPH is formed in genetic engineering bacterium.

3. the sensor-based system according to claim 1 based on flora, which is characterized in that the base containing signal generating module Because the structure of engineering bacteria includes the following steps：

PnpR-PpnpC gene orders pSV- β-galactosidase are inserted by overlap-extension polymerase chain reaction to carry In body, pSVRTCL plasmids are formed, then pSVRTCL plasmids are transformed into genetic engineering bacterium, formed containing signal generating module Genetic engineering bacterium.

4. the sensor-based system according to claim 1 based on flora, which is characterized in that the signal induction module containing OPH Genetic engineering bacterium and the relative scale of genetic engineering bacterium containing signal generating module be 10:1~1:10.

5. the sensor-based system according to claim 1 based on flora, which is characterized in that the signal induction module containing OPH Genetic engineering bacterium it is identical with the gross density of the genetic engineering bacterium containing signal generating module.

6. a kind of application based on sensor-based system described in claim 1 in detecting organophosphorus pesticide.

7. a kind of method of the sensor-based system detection organophosphorus pesticide based on flora, which is characterized in that include the following steps：

Sensor-based system described in claim 1 is suspended in the 2YT fluid nutrient mediums containing X-gal and organophosphor, at 28 DEG C It incubates altogether, the concentration of organophosphor is measured by color intensity.

8. a kind of detection organophosphorus pesticide filter paper based on sensor-based system described in claim 1.

9. it is a kind of it is according to claim 8 detection organophosphorus pesticide filter paper preparation method, which is characterized in that including with Lower step：By the genetic engineering bacterium of the signal induction module described in claim 1 containing OPH and gene work containing signal generating module Journey bacterium is suspended in dry-run protection agent solution, and aseptically drops on filter paper item, cellular regions is formed, then by filter paper item Vacuum freeze drying to get.

10. a kind of application method of detection organophosphorus pesticide filter paper according to claim 8, which is characterized in that including Following steps：Solution to be measured containing organophosphor is dissolved in 2YT fluid nutrient mediums, the culture is then inserted into the cellular regions of filter paper In base, after 28 DEG C are incubated 2 hours, X-gal is added to the cellular regions of filter paper, passes through the dense of Color development strength detection organophosphor Degree.